X chromosome inactivation is the process whereby one of the two X chromosomes in somatic cells of mammalian females is inactivated early in embryogenesis, presumably as a means of dosage compensation between females, with two X chromosomes, and males, with one. While the essential features of X inactivation have been known for over 30 years, the precise molecular, genetic, and developmental details of this lone- range chromosomal control mechanism remain unclear. Over the past few years, substantial progress has been made in the definition and understanding of the various steps involved in X inactivation: initiation early in development through the action of the X inactivation center, promulgation of the inactivation signal and establishment of the inactive state along the chromosome in a strictly cis-limited manner, and maintenance of the various epigenetic states through subsequent cell divisions. A strong candidate for a gene involved in X inactivation, XIST, was described by us in 1991. Since then, significant genetic and developmental evidence has been presented in both humans and mice that supports a role for XIST in the initiation and perhaps establishment of the X inactivation signal. These data provide a framework for further experiments designed to test the hypothesis that the XIST gene, with its regulatory sequences, both controls the initial choice of which X chromosome to inactivate and subsequently establishes a context within the nucleus to distinguish the active from the inactive X chromosome. The experiments described in this application have three specific aims: (1) to determine the nature of the X inactivation center and the role of XIST in initiating X inactivation, by examining the effect(s) of expressing the XIST gene in mouse embryonic stem cells and/or in transgenic mice, by determining the effect(s) of a recently discovered mutation in XIST on the initiation of X inactivation, and by characterizing additional such mutations in families with multiple cases of non-random X inactivation; (2) to determine a developmental context for the establishment of X inactivation, by studying the expression of genes subject to and escaping from X inactivation in mice early in development at the time X inactivation occurs. This is in order to determine whether escape from inactivation is a failure of initiation, establishment, or maintenance of X inactivation; and (3) to examine the chromosomal basis for X inactivation, by studying the distribution of genes on the human and mouse X-chromosomes that are subject to or escape from X inactivation and by using transgenic mice to specifically test the hypothesis that X inactivation is determined at the level of chromosomal domains rather than on an individual gene basis. The proposed experiments should provide a definitive genetic and molecular test of the hypothesis that the XIST gene is, in fact, part of the X inactivation pathway, as well as provide insights into the chromosomal, developmental, and molecular mechanisms by which cis-limited control of X-linked gene expression is achieved.